Post-menopausal and ovariectomized women have a higher risk for cardiovascular disease compared to pre-menopausal women. This decreased risk has been attributed to the beneficial effects of estrogen in premenopausal women and estrogen replacement therapy in post-menopausal woman. Therefore, understanding how estrogen protects against cardiovascular disease is an important problem, not only in women but also in men. Estrogen is a potent vasodilator, and previous studies indicate that estrogen's vasodilatory effect is mediated by endothelium-derived nitric oxide (NO). NO inhibits platelet aggregation, vascular smooth muscle proliferation, and leukocyte adhesion to the vascular wall. Indeed, recent studies suggest that some of estrogen's cardiovascular protective effects are mediated by its stimulatory effects on endothelial nitric oxide synthase (eNOS). Therefore, understanding how estrogen activates eNOS may provide important new insights into some of the rapid molecular actions of estrogen. We have recently reported that estrogen stimulates eNOS activity via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Interestingly, we found that the estrogen receptor (ER) isoform, ERalpha, interacts with the p85alpha regulatory subunit of PI3K in a ligand-dependent manner. Based upon these initial findings, we now propose to determine the subcellular localization of ERa by confocal microscopy; colocalize ERa and p85a using fluorescent resonance energy transfer (FRET); and to identify the putative interaction domain(s) of ERalpha and p85alpha. We then propose to study the physiological significance of this interaction by "knocking in" the mutated ERalpha or p85alpha, which cannot interact with each other, and then to determine whether eNOS activation by estrogen is impaired in these mice. The relevance of this pathway will be further tested in models of vascular injury and ischemic stroke where estrogen and NO have been shown to be protective. The generation of these mice may also be useful in studying the "nuclear" versus "non-nuclear" effects of estrogen in other non-vascular tissues such as bone. The significance of the proposed studies is that by linking the ER to PI3K, a potential critical step in the non-nuclear action of estrogen is suggested and the role of ER is considerably broadened since PI3K is known to mediate diverse cellular functions. These results may explain some of the actions of selective estrogen receptor modulators (SERMs) and provide a therapeutic basis for using estrogens in cardiovascular disease.